Progressive mitochondrial damage and the resulting dysfunction are key contributors to many human diseases. Eukaryotic cells have a multi-layered system for damage prevention and response. Many studies in humans and a variety of model systems have implicated mitochondrial protein degradation as one of the first lines of defense. We have uncovered a system, centered on the evolutionarily conserved Vms1 protein, that promotes the removal of proteins from the mitochondrial outer membrane in a stress- responsive manner. We propose an ambitious inter-disciplinary plan to describe the regulation and function of this protein and its associates in mitochondrial quality control. First, we propose to define the structural and biochemical basis for the stress-responsive localization of Vms1 to mitochondria. This involves both an inhibitory intramolecular interaction and the interaction of Vms1 with a modified sphingolipid. Second, we propose to determine the mechanisms underlying the stress-responsiveness of Vms1 localization, with an initial focus on a lipid-modifying enzyme that migrates to mitochondria under stress. We will also confirm our preliminary data suggesting that these mechanisms are operative in mammalian cells. Finally, we will determine the biochemical and physiological function of Vms1 in mammals using a combination of cell culture and mouse models. This includes the identification of degradation substrates as well as defining the phenotype of a mouse lacking Vms1.